(i) Field of the Invention:
The present invention relates to plant cells and plants, which are genetically modified, wherein the genetic modification leads to the expression in plastids of such plant cells and plants of an enzyme having the activity of a dextransucrase. Furthermore, the present invention relates to means and methods for the manufacture of such plant cells and plants. Plant cells and plants of this type synthesise a modified starch. The present invention therefore also relates to the starch synthesised by the plant cells and plants according to the invention as well as to methods for the manufacture of the starch and to the manufacture of starch derivatives of this modified starch.
(ii) Description of the Related Art:
With respect to the increasing significance which has recently been ascribed to vegetal substances as regenerative sources of raw materials, one of the objects of biotechnological research is to try to adapt vegetal raw materials to the demands of the processing industry. In order to enable the use of regenerative raw materials in as many areas as possible, it is furthermore important to obtain a large variety of substances. Apart from oils, fats and proteins, polysaccharides constitute the essential regenerative raw materials derived from plants. Apart from cellulose, starch maintains an important position among the polysaccharides, being one of the most significant storage substances in higher plants.
Starch is deposited as granules in the chloroplasts of green leaves (transitory starch) and in amyloplasts of tubers, roots and seeds (storage starch) (Kossmann and Llyod 2000).
The polysaccharide starch is a polymer made up of chemically homogeneous basic components, namely the glucose molecules. However, it constitutes a highly complex mixture from various types of molecules which differ from each other in their degree of polymerization and in the degree of branching of the glucose chains. Therefore, starch is not a homogeneous raw material. One differentiates particularly between amylose-starch, a basically non-branched polymer made up of alpha-1,4-glycosidically branched glucose molecules, and amylopectin-starch which in turn is a complex mixture of various branched glucose chains. The branching results from additional alpha-1,6-glycosidic interlinkings.
In plant storage organs, starch biosynthesis takes place within the amyloplast and is the result of different reactions such as synthesis (polymerization of glucosyl residues), rearrangement and degradation, in which various starch synthases (E.C.2.4.1.21), transferases (branching (E.C.2.4.1.18) and disproportionating enzyme (E.C.2.4.1.25)), and hydrolytic enzymes (debranching enzyme (E.C.3.2.1.41)), respectively, play key roles.
In order to enable as wide a use of starch as possible, it seems to be desirable that plants be provided which are capable of synthesizing modified starch which is particularly suitable for various uses. One possibility to provide such plants—apart from breeding methods—is the specific genetic modification of the starch metabolism of starch-producing plants by means of recombinant DNA techniques.
Over the years, several studies have been done aimed at turning the amyloplast into a more versatile polysaccharide factory. For this purpose, several microbial enzymes have been equipped with a plastidial targeting transit, and their influence on starch structure and functionality has been investigated.
Certain bacteria possess an array of enzymes, so-called glucansucrases, which can attach (contiguous) 1,6-linked or 1,3-linked glucosyl residues to maltodextrins. With few exceptions, glucansucrases are extracellular enzymes, which are produced by lactic acid bacteria such as Leuconostoc mesenteroides strains, oral Streptococci, and some species of Lactobacillus and Lactococcus (Robyt 1995; van Geel-Schutten et al. 1999). In addition, they are produced by other bacteria such as some of the Neisseria strains (Hehre et al. 1949). These strains are involved in different processes in nature. Some of the strains colonize the oral cavity of humans and animals and can induce the formation of dental caries. Other strains can invade the throat such as the commensal Neisseria species. Some Lactobacillus species increase the viscosity of fermented milk (de Vuyst and Degeest 1999).
The glucansucrases catalyse the polymerisation of glucose residues from sucrose, which leads to the production of a large variety of α-glucans with different sizes and structures, and composed of diverse linkage types.
Glucansucrases can be classified according to the structure of the glucan formed, and in particular the nature and frequency of the glucosidic linkages synthesized.
Dextransucrase (DSR) (E.C.2.4.1.5) synthesizes a glucan, called dextran, mainly composed of α-(1→6) glucosyl residues in the main linear chain and branched by variable proportions of α-(1→2), α-(1→3) or α-(1→4) linkages (Jeanes et al., 1954; Sidebotham, 1975; Robyt, 1995).
Biosynthesis of dextrans is mediated by Lactobacillus, Leuconostoc, and Streptococcus bacteria in the presence of sucrose.
Nucleic acid sequences encoding dextransucrases are well known in the art and numerous different sequences have been identified for numerous different dextransucrases (GenBank Database).
Dextran produced by Leuconostoc mesenteroides NRRL B-512F is water-soluble, and consists of 95% α-(1→6) linkages in the main chain and 5% α-(1→3) side chains (van Cleve et al., 1956). Its biosynthesis is mediated by a dextransucrase DSR-S (EC 2.4.1.5), which is a 1,527 amino-acid glucosyltransferase (Wilke-Douglas et al., 1989; Monchois et al., 1997). Its catalytic properties can be summarized as follows: after cleavage of sucrose, the glucose residue can be transferred to a growing dextran chain by the so-called two-site insertion mechanism, or to acceptor molecules (Robyt, 1995; Monchois et al., 1999).
The sequence of the Dsr-S gene from L. mesenteroides NRRL B-512F has been reported in WO 89/12386 and by Quirasco et al (1999); GenBank (Accession AJ271107).
Most glucansucrases share a common structure composed of four different regions: a signal peptide, a variable region, a catalytic domain, and a glucan-binding domain (GBD). (Monchois et al., 1999, FEMS Microbiology Letters 177, 243-248; Monchois et al., 1999, FEMS Microbiology Reviews 23, 131-151).
The signal peptide consists of 35-38 amino acids and is responsible for secretion of the sucrases, when expressed by their natural bacterial hosts. The signal peptide is followed by a variable region of 140-261 amino acids. Janecek et al. (2000) showed that conserved, long repeat elements (A-like repeats) are present in the downstream part of this region of dextransucrases of Leuconostoc mesenteroides NRRL B-512F (DSR-S), NRRL B-1299 (DSR-B). It has been hypothesized that these repeats can play a role in glucan binding. Nevertheless, this region does not seem to be essential for enzyme activity because DSR-A, which is produced by Leuconostoc mesenteroides NRRL B-1299 and catalyses the formation of a polymer containing between 27 and 35% of α-(1→2) branched linkages in addition to α-(1→6) ones (Robyt et al, 1978), does not possess this region and is still active.
The catalytic domain is composed of about 900 amino acids and is highly conserved within the Leuconostoc and Streptococcus species (MacGregor et al. 1996). The catalytic domain is also called the sucrose-binding domain because it contains a catalytic triad of aspartic and glutamic acid residues that play an important role in binding and cleavage of sucrose molecules.
The glucan-binding domain is covering about 500 amino acids, and is composed of repeats named A, B, C, D that are defined by a consensus sequence (Monchois et al 1998, 1999). Nevertheless, the number and organization of these repeats is variable within glucansucrases, and it has been shown that the minimum number of these repeated units necessary to ensure glucan binding properties is different according to the enzymes, and more particularly is different for enzymes producing a soluble glucan than for those producing an insoluble one (Monchois et al., 1999)
The elongation of glucan chains by glucansucrases is quite different compared to that by starch synthases. First, the preferred substrate is sucrose instead of ADP-Glucose. Second, the glucose residues are added to the reducing end of a growing glucan chain by a so-called two-site insertion mechanism (Robyt 1995).
In addition, the branching of glucans does not take place by means of a branching enzyme as in starch biosynthesis, but by a so-called acceptor reaction catalyzed by the glucansucrases themselves (Robyt, 1995). The glucansucrase is thought to contain an acceptor-binding site that can bind acceptor molecules such as the nascent glucan chains or maltodextrins (Su and Robyt, 1994).
The efficiency to catalyse acceptor reactions, particularly with starch polymers or maltodextrins is nevertheless unpredictable, as the structure-function relationships underlying the acceptor reaction are not understood and is poorly documented. It seems nevertheless that the relative acceptor efficiency depends on the size of the acceptor molecules (Fu et al. 1990), and it is uncertain that amylopectine and amylose may be acceptor molecules for glucansucrases.
Starch polymer modification has been achieved by targeting the Escherichia coli glycogen synthase (GLGA) and the glycogen branching enzyme (GLGB) to the potato amyloplast (Shewmaker et al. 1994; Kortstee et al. 1996). In both cases, the natural balance of chain elongation and branching was disturbed, resulting in starch granules with altered physical properties, and with more heavily branched polymers.
Attachment of novel glycosyl residues to starch polymers has also been an objective. For this purpose, a Bacillus subtilis levansucrase (E.C.2.4.1.10) was introduced in potato tuber amyloplasts (Gerrits et al. 2001). Levansucrase can polymerize the fructose moiety of the donor substrate sucrose into a high molecular weight fructan. Nevertheless, the starch yield was severely compromised and the starch morphology was dramatically altered.
It has also been tried to convert starch in planta into high-value cyclic oligosaccharides, which can accommodate hydrophobic substances in their apolar cavity and can be used in various food and pharmaceutical applications. A cyclodextrin glycosyltransferase (CGTase; E.C.2.4.1.19) from Klebsiella pneumoniae was introduced into potato amyloplasts (Oakes et al. 1991) for cyclodextrin production. Only 0.01% of the endogenous starch was converted to the desired product, and this product was difficult to recover from the plant material.
These examples demonstrate that bacterial enzymes can be potentially powerful tools for starch modification, but that their performance in the plant are unpredictable beforehand (Kok-Jacob A. et al, 2003).